Mycobacterium Neoaurum-Derived Steroid C27-monooxygenase and Application Thereof

ABSTRACT

The present invention discloses a  Mycobacterium neoaurum -derived steroid C27-monooxygenase and an application thereof, which belong to the technical fields of genetic engineering and enzyme engineering. By the method of gene knockout and intensive expression, the present invention screens out three isoenzymes of a key enzyme SMO in the process of degrading sterol side chains from  Mycobacterium neoaurum . The three isoenzymes are intensively expressed respectively in the  Mycobacterium neoaurum  for the high yield of androsta-1,4-diene-3,17-dione (ADD), the yield of ADD is increased remarkably, wherein the effect of SMO2 is most remarkable. By overexpressing SMO2, the final ADD yield is increased from 5.2 g·L. −1  to 7.3 g·L. −1 . The present invention provides a helpful guidance for the industrialization of the microbial fermentation method for increasing the ADD yield.

TECHNICAL FIELD

The disclosure herein relates to the field of genetic engineering and enzyme engineering, and more particularly relates to a mycobacterium neoaurum-derived steroid C27-monooxygenase and an application thereof.

BACKGROUND

Because of a variety of physiological functions, steroid hormone drugs play a very important role in regulating the body, and therefore have been widely applied clinically. Steroid hormone drugs have been used widely to resist tumors, inflammation, bacteria, viruses, hormones, allergy, etc. In addition, various sex hormone drugs are main drugs for treating sexual organ degradation and gynecological diseases, and are main ingredients of oral contraceptives. Moreover, steroid hormone drugs can also serve as active ingredients of anti-obesity drugs for preventing coronary heart disease, and as HIV integrase inhibitors for preventing HIV infection and treating AIDS, etc. As important intermediates of the steroid hormone drugs, androsta-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) can almost synthesize all steroid hormone drugs, and play an important role in the industrialized production of steroids.

At present, there are two main approaches to the preparation of AD and ADD: in one approach, diosgenin is extracted from wide Chinese herbal medicine plants, such as Dioscorea Zingiberensis C. H. Wright and Dioscorea althaeoides R. Knuth, and chemical synthesis is then carried out. However, this approach has the problems of high production cost, complex process, severe environment pollution, etc; in the other approach, with zoosterol and phytosterol existing abundantly in nature as materials, by means of the microbial fermentation technique, sterol side chains are degraded selectively, and thereby a target product is obtained. The microbial method has the advantages of low cost, environment-friendliness, etc., and is more and more valued by people. At present, when conducting steroid drug preparation, the advanced countries in the world mostly adopt the zoosterol and the phytosterol as starting materials to carry out microbial transformation, and obtain steroid drug intermediates before proceeding with preparation.

Many scholars have conducted a lot of researches on the synthesis of AD/ADD by the microbial transformation of sterols, wherein mycobacteria are one of excellent strains for the production of AD/ADD. It has been reported that the metabolic pathway of the synthesis of AD/ADD by the microbial transformation of sterols requires a large number of enzymes, but which enzymes participate have not yet been thoroughly determined in researches. The progress of current researches is that only the enzyme in the first step of sterol transformation, which is cholesterol oxidase, and the enzyme in the last step, which is 3-ketosteroid-Δ¹-dehydrogenase, have been identified and proved. Other enzymes in the approach have not yet been identified and applied.

Therefore, it is necessary to make in-depth research on key enzymes in the process of synthesizing AD/ADD by utilizing mycobacteria to transform sterols and the identification of their functions in order to develop a method capable of promoting mycobacteria to transform sterols to synthesize AD/ADD on this basis.

SUMMARY

In order to solve the above-mentioned problem, by the method of gene knockout and complementation, the present invention has screened out three isoenzymes (SMO1, SMO2, SMO3) of steroid C27-monooxygenase (SMO) as a key enzyme in the process of degrading sterol side chains from mycobacterium neoaurum for the first time. By respectively intensively expressing the three isoenzymes in the mycobacterium neoaurum for the high yield of androsta-1,4-diene-3,17-dione (ADD), ADD yields are all increased obviously, wherein the effect of the intensive expression of SMO2 in increasing the yield of ADD is more remarkable. Both the function identification and application of the steroid C27-monooxygenase in the mycobacterium neoaurum are reported for the first time, and recombinant bacteria can significantly increase the yield of ADD. The final ADD yield of the strain intensively expressing the gene SMO2 is increased from 5.2 g·L⁻¹ to 7.3 g·L⁻¹.

The first objective of the present invention is to provide a steroid C27-monooxygenase, and the amino acid sequence of the steroid C27-monooxygenase is shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.

The second objective of the present invention is to provide a recombinant mycobacterium neoaurum for increasing the yield of ADD, and the recombinant bacteria are mycobacterium neoaurum overexpressing a steroid C27-monooxygenase gene; and the steroid C27-monooxygenase gene is any one or two or more of genes Smo1, Smo2 and Smo3.

In one embodiment of the present invention, the amino acid sequence of the steroid C27-monooxygenase gene Smo1 is shown as SEQ ID NO. 4, and the nucleotide sequence is shown as SEQ ID NO. 1.

In one embodiment of the present invention, the amino acid sequence of the steroid C27-monooxygenase gene Smog is shown as SEQ ID NO. 5, and the nucleotide sequence is shown as SEQ ID NO. 2.

In one embodiment of the present invention, the amino acid sequence of the steroid C27-monooxygenase gene Smo3 is shown as SEQ ID NO. 6, and the nucleotide sequence is shown as SEQ ID NO. 3.

In one embodiment of the present invention, the recombinant mycobacterium neoaurum overexpresses the steroid C27-monooxygenase genes with mycobacterium neoaurum as an original strain.

In one embodiment of the present invention, the original strain can be any one of the following strains: Mycobacterium neoaurum ATCC 25795, Mycobacterium neoaurum ZJUVN (CGMCC 5477), Mycobacterium neoaurum NwIB-01 (CCTCC M 209094), Gordonia neofelifaecis NRRL B-59395 (CCTCC AB-209144), Arthrobacter simplex (TCCC 11037), Mycobacterium fortuitum subsp. fortuitum (MTCC 929) or Mycobacterium neoaurum JC-12.

Mycobacterium neoaurum ATCC 25795 has been disclosed by Yao K, Ku L-Q, Wang F-Q, Wei D-Z (2014) in the document of Characterization and engineering of 3-ketosteroid-Δ1-dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9α-hydroxy-4-androstene-3,17-dione through the catabolism of sterols. Metab Eng 24(0):181-191 doi:http:// dx.doi.org/10.1016/j.ymben.2014.05.005.

Mycobacterium neoaurum ZJUVN (CGMCC 5477) has been disclosed by Zhang X-y, Peng Y, Su Z-r, Chen Q-h, Ruan H, He G-q (2013) in the document of Optimization of biotransformation from phytosterol to androstenedione by a mutant Mycobacterium neoaurum ZJUVN-08. Journal of Zhejiang University SCIENCE B 14(2):132-143 doi:10.1631/jzus.B1200067.

Mycobacterium neoaurum NwIB-01 (CCTCC M 209094) has been disclosed by Wei W, Fan S-Y, Wang F-Q, Wei D-Z (2014) in the document of Accumulation of androstadiene-dione by overexpression of heterologous 3-ketosteroid Δ1-dehydrogenase in Mycobacterium neoaurum NwIB-01. World J Microbiol Biotechnol 30(7):1947-1954 doi:10.1007/s11274-014-1614-3.

Gordonia neofelifaecis NRRL B-59395 (CCTCC AB-209144) has been disclosed by Liu Y, Chen G, Ge F, Li W, Zeng L, Cao W (2011) in the document of Efficient biotransformation of cholesterol to androsta-1,4-diene-3,17-dione by a newly isolated actinomycete Gordonia neofelifaecis. World J Microbiol Biotechnol 27(4):759-765 doi:10.1007/s11274-010-0513-5.

Arthrobacter simplex (TCCC 11037) has been disclosed by Wang M, Zhang L T, Shen Y B, Ma Y H, Zheng Y, Luo J M (2009) in the document of Effects of hydroxypropyl-β-cyclodextrin on steroids 1-en-dehydrogenation biotransformation by Arthrobacter simplex TCCC 11037. J Mol Catal, B Enzym 59(1-3):58-63 doi:h p:// dx.doi.org/10.1016/j.molcatb.2008.12.017.

Mycobacterium fortuitum subsp.fortuitum (MTCC 929) has been disclosed by Galla V, Banerjee T, Patil S (2010) in the document of Bioconversion of soysterols to androstenedione by Mycobacterium fortuitum subsp.fortuitum NCIM 5239, a mutant derived from total sterol degrader strain. J Chem Technol Biotechnol 85(8):1135-1141 doi:10.1002/jctb.2410.

Mycobacterium neoaurum JC-12 has been disclosed by Shao M L, Zhang X, Rao Z M, Xu M J, Yang T W, Li H, Xu Z1-I (2015) in the document of Enhanced production of androsta-1,4-diene-3,17-dione by Mycobacterium neoaurum JC-12 using three-stage fermentation strategy. PLoS ONE 10(9): e0137658.

In one embodiment of the present invention, with regard to overexpression, specifically, the steroid C27-monooxygenase genes are connected to a plasmid pMV261, so that recombinant plasmids are obtained, and the recombinant plasmids are then transformed into the original strain.

The third objective of the present invention is to provide a method for increasing the yield of ADD by overexpressing a steroid C27-monooxygenase gene, the method utilizes the recombinant mycobacterium neoaurum disclosed by the present invention as a production strain to fermentatively produce ADD; the recombinant mycobacterium neoaurum overexpresses the steroid C27-monooxygenase gene; and the steroid C27-monooxygenase gene is any one or two or more of genes Smo1, Smo2 and Smo3.

In one embodiment of the present invention, the amino acid sequence of the steroid C27-monooxygenase gene is shown as SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

In one embodiment of the present invention, fermentative production adopts phytosterol or/and cholesterol as a substrate.

In one embodiment of the present invention, the ingredients of a fermentation medium for fermentation are: 20 g·L⁻¹ cholesterol, 20 g·L⁻¹ glucose, 10 g·L⁻¹ peptone, 6 g·L⁻¹ beef extract, 3 g·L⁻¹ K₂HPO₄, 0.5 g·L⁻¹ MgSO₄·7H₂O, 5×10⁻⁴ g·L⁻MnCl₂·4H₂O and 60 g·L⁻¹ hydroxypropyl- β-cyclodextrin, and pH is 7.5.

In one embodiment of the present invention, in fermentative production, fermentation is carried out under the conditions of 30° C. and 160 rpm for 168 h.

The fourth objective of the present invention is to provide ADD produced from the recombinant bacteria.

The fifth objective of the present invention is to provide an application of the recombinant bacteria or the steroid C27-monooxygenase genes in the preparation of a drug.

In one embodiment of the present invention, the drug is an anti-tumor, anti-inflammation, antibacterial, antiviral, anti-hormone or antiallergic drug.

In one embodiment of the present invention, the drug is a drug for treating sexual organ degeneration, a drug for gynecological diseases, an anti-obesity drug, a drug for preventing coronary heart disease, an HIV integrase inhibitor or etc.

(1) The present invention screens out the three isoenzyme genes of the steroid C27-monooxygenase in the mycobacterium neoaurum by a genetic engineering means, and identifies the enzymes in the second step of the sterol transformation process based on the steroid C27-monooxygenase (SMO) by means of the method of gene knockout and knocked-out gene complementation. The present invention proves the functions of the three isoenzymes in the process of sterol metabolism, determines their key role in the process of sterol transformation, and verifies they are key enzymes in the process of synthesizing AD/ADD by utilizing mycobacteria to transform sterols.

(2) On the basis of determining the key enzymes, the present invention applies them in the process of synthesizing AD/ADD by sterol transformation in order to increase the yield of ADD, and has discovered that the effect of SMO2 in increasing the yield of ADD is most remarkable. Ultimately, by overexpressing SMO2, the yield of ADD is increased from 5.2 g·L⁻¹ to 7.3 g·L⁻¹, increased by 40.4 percent in comparison with the original bacteria. The present invention provides a helpful guidance for the industrialization of the microbial fermentation method for increasing the yield of ADD.

DETAILED DESCRIPTION

Main reagents: Phytosterol (soyasterol≥95%) was purchased from Lilly Biotechnology (Huzhou) Co., Ltd, and ADD was purchased from American SIGMA company.

HPLC analysis of ADD: ADD had characteristic absorption peaks under an ultraviolet wavelength of 254 nm, so the HPLC method was adopted to determine product concentration. Chromatographic conditions: chromatographic column: DimosoilC18 (5 μl, 250 mm×4.6 mm); mobile phase: methanol-water (V/V=70:30); detector: UV Detector; detection wavelength: 254 nm; column temperature: 30° C.; sample amount: 104; and flow velocity: 1.0 ml·min⁻¹.

Example 1: Construction of SMO Knockout Strains and Corresponding Complementation Strains

By querying the whole genome information of Mycobacterium neoaurum, three SMO isoenzymes were screened out. With the Mycobacterium neoaurum with SMO enzyme activity in a lab as an original strain and its chromosome as a template, the PCR measure was utilized to obtain the genes of the three enzymes. By means of the design of gene knockout primers, the PCR measure was utilized to obtain knocked-out genes, which were connected to a mycobacterium knockout plasmid p2NIL, so that knockout plasmids were constructed, and after the construction was verified successful by PCR, the knockout plasmids were transformed into the Mycobacterium neoaurum. With cholest-4-en-3-one as a substrate, the degradation of the substrate by knockout strains was tested. On the basis of the knockout strains, knocked-out gene complementation strains were constructed, that was, by designing primers, the PCR measure was utilized to amplify complete SMO genes, which were connected to an integration vector pMV306, so that integrative plasmids were constructed, and after the construction was verified successful by PCR, the integrative plasmids were transformed into the corresponding knockout strains, so that the knocked-out gene complementation strains were constructed; and under the same conditions, with cholest-4-en-3-one as a substrate, the degradation of the substrate by the complementation strains was tested. A test result indicated that the ability of the gene knockout strains in degrading cholest-4-en-3-one was impeded, while the knocked-out gene complementation strains decreased such an impeding effect to a certain degree. The present invention proved in the end that the three enzymes SMO were key enzymes in the process of sterol degradation.

A specific construction method for the combinant strains was as follows:

(1) Cloning the Complete Sequence of a Steroid C27-Monooxygenase (SMO) Gene

According to the whole genome sequence (NC-023036) of Mycobacterium neoaurum VKM Ac-1815D published by the website GENBANK, three SMO genes (Smo1, Smo2, Smo3) were found out, and according to specific gene sequences, corresponding gene primers ere designed. With prepared Mycobacterium neoaurum chromosome DNA as a template, corresponding complete gene sequences were amplified out by PCR.

PCR reaction system: 5 μL of 10×ExTaq Buffer; 4 μL of dNTP; 1 μL of template DNA; 0.5 μL of upstream primer; 0.5 μL of downstream primer; 1 μL of ExTaq enzyme; and ddH₂O accounting for the rest of a total volume of 50 μL. PCR reaction condition: 94° C. 5 min; 94 ° C. 30 s; 65° C. 45 s; 72° C. 90 s; 35 times of cycling; 72° C. 10 min; and 12° C. 10 min.

Referring to the instructions of a gel extraction kit of Shanghai Generay Biotech Co., Ltd, a PCR product was recycled. The gel extraction product was connected to pMD18-T vector overnight according to a certain proportion, E. coli JM109 competent cells were transformed, an ampicillin resistant plate was utilized to screen recombinant bacteria, recombinant plasmids underwent restriction enzyme digestion to release gene bands, the sizes of which were about 2.7 kb and 1.3 kb, this indicated that recombinant plasmid construction was successful, and the recombinant plasmids were named as pMD18-T-Smo1, pMD18-T-Smo2 and pMD18-T-Smo3.

(2) Constructing Mycobacterium neoaurum Gene Knockout Plasmids

Specific primers were designed, and by way of PCR, 300 bp sequences were amplified out from the upstream of Smo1, Smo2 and Smo3 and 450 bp sequences were amplified out from the downstream. The gene sequences amplified from the upstream and the downstream were mixed as a template, and the upstream primers of the upstream 300 bp sequences and the downstream primers of the 450 bp sequences were utilized to amplify out 750 bp sequences. The amplified sequences and a knockout plasmid p2NIL underwent double-restriction enzyme (KpnI and Hind III) digestion, gel extraction and purification were conducted, T4 DNA ligase was connected to two segments overnight, conjugates underwent heat shock to transform E. coli JM109 competent cells after overnight, and a kanamycin resistant plate was utilized to screen positive transformants. Transformant plasmids were extracted, and the construction of recombinant plasmids was verified successful by restriction enzyme digestion. Single-restriction enzyme (Pac I) digestion was utilized to cut off a selectable marker gene cassette in a plasmid pGOAL19 and inserted it into the restriction enzyme (Pac I) digestion sites of the constructed recombinant plasmids, so that knockout plasmids p2N-ΔSmo1, p2N-ΔSmo2 and p2N-ΔSmo3 were constructed. The construction of the knockout plasmids was verified successful by PCR.

(3) Constructing Mycobacterium neoaurum Knocked-Out Gene Complementation Plasmids

A plasmid pMV306 was used for the complementation of knocked-out genes. Double-restriction enzyme (Xba Ida I) digestion was utilized to cut off the corresponding gene segments in the recombinant plasmids p261-Smo1, p261-Smo2 and p261-Smo3 along with heat shock promoters hsp60 and connected them onto corresponding restriction enzyme (pMV306) digestion sites, so that knocked-out gene complementation plasmids p306-Smo1, p306-Smo2 and p306-Smo3 were constructed.

(4) Transforming the Recombinant Plasmids into the Mycobacterium neoaurum by an Electrotransformation Method

A. The successfully constructed recombinant plasmids were transformed into the Mycobacterium neoaurum by using an electrotransformation method. The improved Gordhan and Parish method was adopted as the transformation method.

B. Positive transformants of the recombinant strains M. neoaurum were screened;

Colonies which grow out on plates with corresponding antibiotic pressures were chosen, flasks were shaken for fermentation, and the plasmids were extracted to be verified by restriction enzyme digestion.

Example 2: Construction of Recombinant Strains Intensively Expressing SMO

A plasmid pMV261 was utilized to overexpress the gene in Mycobacterium neoaurum. Sac I and Hind III were utilized to carry out double-restriction enzyme digestion on pMD18-T-Smo1, BamH I and EcoR I were utilized to respectively carry out double-restriction enzyme digestion on pMD18-T-Smo2 and pMD18-T-Smo3, meanwhile, a corresponding restriction enzyme digestion site was utilized to carry out restriction enzyme digestion on the plasmid pMV261, gel extraction and purification were carried out on the corresponding gene segments and the plasmid pMV261 segment, T₄ DNA ligase connected the two segments overnight, conjugates underwent heat shock to transform E. coli JM109 competent cells after overnight, and a kanamycin resistant plate was utilized to screen positive transformants. Transformant plasmids were extracted, the construction of recombinant plasmids p261-Smo1, p261-Smo2 and p261-Smo3 was verified successful by restriction enzyme digestion, the successfully constructed recombinant plasmids were electrotransformed respectively into Mycobacterium neoaurum JC-12 for the high yield of ADD (Mycobacterium neoaurum JC-12 had been disclosed by Shao M L, Zhang X, Rao Z M, Xu Mi, Yang T W, Li H, Xu Z H (2015) in the document of Enhanced production of androsta-1,4-diene-3,17-dione by Mycobacterium neoaurum JC-12 using three-stage fermentation strategy. PLoS ONE 10(9): e0137658), and thereby recombinant strains JC-12_(S1), JC-12_(S2) and JC-12_(S3) were obtained respectively.

Example 3: Increase of ADD Yield by Recombinant Strains Intensively Expressing SMO

After being activated in a seed medium, the recombinant strains JC-12_(S1), JC-12_(S2) and JC-12_(S3) were inoculated into a fermentation medium according to an inoculation amount of 5%, fermentation was carried out with 20 g·L⁻¹ cholesterol as a substrate under the conditions of 30° C. and 160 rpm for 168 h, and a fermentative transformation experiment was conducted. The seed medium consisted of 10 g·L⁻¹ glucose, 10 g·L⁻¹ peptone, 6 g·L⁻¹ beef extract and 10 g·L⁻¹ NaCl, and pH was 7.5. The fermentation medium consisted of 20 g·L⁻¹ cholesterol, 20 g·L⁻¹ glucose, 10 g·L⁻¹ peptone, 6 g·L⁻¹ beef extract, 3 g·L⁻¹ K₂HPO₄, 0.5 g·L⁻¹ MgSO₄·7H₂O, 5×10−4 g·L⁻¹ MnCl₂4H₂O and 60 g·L⁻¹ hydroxypropyl-β-cyclodextrin, and pH was 7.5.

The change of the ADD yield was detected. A result indicated that after 168 h of fermentative transformation, the ADD yield of the recombinant bacteria JC-12_(S2) overexpressing

SMO2 was increased most remarkably, and increased from 5.2 g·L⁻¹ Mycobacterium neoaurum JC-12 as the original strain to 7.3 g·L⁻¹. In addition, the ADD yields of the recombinant bacteria JC-12_(S1) and JC-12_(S3) were increased respectively to 6.5 g·L⁻¹ and 6.1 g·L⁻¹. 

1. Recombinant Mycobacterium neoaurum with an increased yield of ADD, wherein the recombinant Mycobacterium neoaurum comprises an overexpression of a steroid C27-monooxygenase gene; and the steroid C27-monooxygenase gene is selected from the group consisting of Smo1 gene, Smo2 gene and Smo3 gene.
 2. The recombinant Mycobacteriurn neoaurum according to claim 1, characterized in that the amino acid sequences of the steroid C27-monooxygenase genes Smo1, Smo2 and Smo3 are set forth respectively in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
 3. The recombinant Mycobacterium neoaurum according to claim 1, characterized in that with regard to the overexpression, the recombinant Mycobacterium neoaurum comprises a recombinant plasmid comprising the steroid C27-monooxygenase gene.
 4. A method for increasing the yield of ADD by overexpressing a steroid C27-monooxygenase gene, comprising using the recombinant Mycobacterium neoaurum of claim 1 as a production strain; overexpressing the steroid C27-monooxygenase gene in the recombinant Mycobacterium neoaurum; and fermentatively producing ADD.
 5. The method according to claim 4, characterized in that the amino acid sequence of the steroid C27-monooxygenase gene Smo1 gene is set forth in SEQ ID NO.4, the amino acid sequence of the steroid C27-monooxygenase gene Smo2 gene is set forth in SEQ ID NO. 5, and the amino acid sequence of the steroid C27-monooxygenase gene Smo3 gene is set forth in SEQ ID NO.6,
 6. The method according to claim 4, comprising using phytosterol or cholesterol or both as a substrate.
 7. (canceled)
 8. A method of preparing a drug, comprising using the recombinant Mycobacterium neoaurum of claim 1 as a production strain; overexpressing the steroid C27-monooxygenase gene in the recombinant Mycobacterium neoaurum; fermentatively producing ADD; and preparing a drug comprising the ADD.
 9. The method according to claim 8, characterized in that the drug is an anti-tumor, anti-inflammation, antibacterial, antiviral, anti-hormone or antiallergic drug.
 10. The method according to claim 8, characterized in that the drug is a drug for treating sexual organ degeneration, a drug for gynecological diseases, an anti-obesity drug, a drug for preventing coronary heart disease or an HIV integrase inhibitor. 